Compositions of the SOC/CRAC calcium channel protein family

ABSTRACT

Nucleic acids encoding SOC/CRAC calcium channel polypeptides, including fragments and biologically functional variants thereof and encoded polypeptides are provided. The nucleic acids and polypeptides disclosed herein are useful as therapeutic and diagnostic agents. Agents that selectively bind to the foregoing polypeptides and genes also are provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §120 or 35 U.S.C.§365(c) of PCT International application PCT/US99/29996 designating theUnited States of America, and filed Dec. 20, 1999, of which thisapplication is a national stage filing under 35 U.S.C. §371, waspublished under PCT Article 21(2) in English.

Application number PCT/US99/29996 claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application 60/114,220, filed Dec. 30, 1998;60/120,018, filed Jan. 29, 1999; and 60/140,415, filed Jun. 22, 1999.

FIELD OF THE INVENTION

This invention relates to nucleic acids coding for a novel family ofcalcium channel polypeptides, the encoded polypeptides, unique fragmentsof the foregoing, and methods of making and using same.

BACKGROUND OF THE INVENTION

Calcium channels are membrane-spanning, multi-subunit proteins thatfacilitate the controlled transport (“flux”) of Ca²⁺ ions into and outof cells. Cells throughout the animal kingdom, and at least somebacterial, fungal and plant cells, possess one or more types of calciumchannels. In general, “excitable” cells, such as neurons of the centralnervous system, peripheral nerve cells, and muscle cells, includingthose of skeletal muscles, cardiac muscles, and venous and arterialsmooth muscles, possess voltage-dependent calcium channels. In avoltage-dependent calcium channel, the transport of Ca²⁺ ions into andout of the cells requires a certain minimal level of depolarization (thedifference in potential between the inside of the cell bearing thechannel and the extracellular environment) with the rate of Ca²⁺ cellflux dependent on the difference in potential. In “non-excitable” cells,calcium influx is thought to occur predominantly in response to stimuliwhich cause the release of calcium from intracellular stores. Thisprocess, termed store operated calcium influx, is not well understood.

Characterization of a particular type of calcium channel by analysis ofwhole cells is complicated by the presence of mixed populations ofdifferent types of calcium channels in the majority of cells. Althoughsingle-channel recording methods can be used to examine individualcalcium channels, such analysis does not reveal information related tothe molecular structure or biochemical composition of the channel.Furthermore, in this type of analysis, the channel is isolated fromother cellular constituents that might be important for the channel'snatural functions and pharmacological interactions. To study the calciumchannel structure-function relationship, large amounts of pure channelprotein are needed. However, acquiring large amounts of pure protein isdifficult in view of the complex nature of these multisubunit proteins,the varying concentrations of calcium channel proteins in tissuesources, the presence of mixed populations of calcium channel proteinsin tissues, and the modifications of the native protein that can occurduring the isolation procedure.

SUMMARY OF THE INVENTION

The invention is based on the identification of a novel family ofcalcium channel polypeptides and the molecular cloning and partialcharacterization of a novel member of this family that is expressedpredominantly in human hematopoietic cells, liver, and kidney. Thisnewly identified family of calcium channel polypeptides is designated,“SOC” or “CRAC” or “ICRAC”, for Store Operated Channels or CalciumRelease Activated Channels. Although not wishing to be bound to anyparticular theory or mechanism, it is believed that the SOC/CRAC calciumchannel polypeptides are transmembrane polypeptides that modulate Ca²⁺flux “into” and “out of” a cell, for example, in certain instances theymay be activated upon depletion of Ca²⁺ from intracellular calciumstores, allowing Ca²⁺ influx into the cell. Accordingly, thecompositions disclosed herein are believed to be useful for modulatingcalcium transport into and out of such intracellular stores and for thetreatment of disorders that are characterized by aberrant calciumtransport into and out of such intracellular stores. In particular, webelieve that the SOC/CRAC calcium channel polypeptides disclosed hereinplay an important role in the influx of extracellular calcium bymediating the refilling of intracellular calcium stores following theirdepletion. Accordingly, we believe that the compositions for expressingfunctional SOC/CRAC calcium channel polypeptides in cells, as disclosedherein, are useful for treating patients having conditions that arecharacterized by reduced extracellular calcium influx into theirSOC/CRAC-expressing cells. Additionally, the compositions of theinvention are useful for delivering therapeutic and/or imaging agents tocells which preferentially express SOC/CRAC calcium channel polypeptidesand, in particular, for delivering such agents to hematopoietic cells,liver, heart, spleen, and kidney to modulate proliferation and growth ofthese cells. Moreover, in view of the importance of cellular calciumlevels to cell viability, we believe that SOC-2/CRAC-1, SOC-3/CRAC-2,and SOC-4/CRAC-3 as disclosed herein, and/or other members of theSOC/CRAC family of calcium channel polypeptides, represent an idealtarget for designing and/or identifying (e.g., from molecular libraries)small molecule inhibitors that block lymphocyte proliferation, as wellas other binding agents that selectively bind to SOC/CRAC polypeptidesto which drugs or toxins can be conjugated for delivery to SOC/CRACpolypeptide expressing cells.

The invention is based, in part, on the molecular cloning and sequenceanalysis of the novel SOC/CRAC calcium channel molecules disclosedherein (also referred to as a “SOC-2/CRAC-1 molecule,” a “SOC-3/CRAC-2molecule,” and/or “SOC-4/CRAC-3 molecule”) that are predominantlyexpressed in human hematopoietic cells, liver, spleen, heart, and kidney(SOC-2/CRAC-1), kidney and colon (SOC-3/CRAC-2), and prostate(SOC-4/CRAC-3 molecule). As used herein, a “SOC/CRAC molecule” embracesa “SOC/CRAC calcium channel nucleic acid” (or “SOC/CRAC nucleic acid”)and a “SOC/CRAC calcium channel polypeptide” (or “SOC/CRACpolypeptide”). Homologs and alleles also are embraced within the meaningof a SOC/CRAC calcium channel molecule.

According to one aspect of the invention, isolated SOC/CRAC nucleicacids which code for one or more member(s) of the SOC/CRAC family ofcalcium channel polypeptides or unique fragments thereof are provided.The isolated nucleic acids refer to one or more of the following:

(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and which code for aSOC/CRAC polypeptide;

(b) deletions, additions and substitutions of (a) which code for arespective SOC/CRAC polypeptide;

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b) or (c).

The invention in another aspect provides an isolated nucleic acidmolecule selected from the group consisting of (a) a unique fragment ofa nucleic acid molecule selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:29, and SEQ ID NO:31, (b) complements of (a), provided thatthe unique fragment includes a sequence of contiguous nucleotides whichis not identical to any sequence selected from a sequence groupconsisting of (1) sequences having the SEQ ID NOs or GenBank accessionnumbers of Table I, (2) complements of (1), and (3) fragments of (1) and(2).

According to yet another aspect of the invention, isolated SOC/CRACpolypeptides are provided. The isolated SOC/CRAC polypeptide moleculesare encoded by one or more SOC/CRAC nucleic acid molecules of theinvention. Preferably, the SOC/CRAC polypeptide contains one or morepolypeptides selected from the group consisting of the polypeptideshaving SEQ ID NOs 2, 4, 6, 8, 24, 26, 28, 30, and 32. In otherembodiments, the isolated polypeptide may be a fragment or variant ofthe foregoing SOC/CRAC polypeptide molecules of sufficient length torepresent a sequence unique within the human genome, and identifyingwith a polypeptide that functions as a calcium channel, provided thatthe fragment excludes a sequence of contiguous amino acids identified inTable II, and/or excludes a sequence of contiguous amino acids encodedfor by a nucleic acid sequence identified in Table I. In anotherembodiment, immunogenic fragments of the polypeptide molecules describedabove are provided.

According to another aspect of the invention, isolated SOC/CRAC bindingagents (e.g., polypeptides) are provided which selectively bind to aSOC/CRAC molecule (e.g., a SOC/CRAC polypeptide encoded by the isolatednucleic acid molecules of the invention). Preferably, the isolatedbinding agents selectively bind to a polypeptide which comprises thesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQID NO:30, and SEQ ID NO:32, or unique fragments thereof. In thepreferred embodiments, the isolated binding polypeptides includeantibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fd andantibody fragments which include a CDR3 region which binds selectivelyto a SOC/CRAC polypeptide). Preferably, the antibodies for humantherapeutic applications are human antibodies.

According to another aspect of the invention, a pharmaceuticalcomposition containing a pharmaceutically effective amount of anisolated SOC/CRAC nucleic acid, an isolated SOC/CRAC polypeptide, or anisolated SOC/CRAC binding polypeptide in a pharmaceutically acceptablecarrier also is provided. The pharmaceutical compositions are useful inaccordance with therapeutic methods disclosed herein.

According to yet another aspect of the invention, a method for isolatinga SOC/CRAC molecule is provided. The method involves:

a) contacting a SOC/CRAC nucleic acid or a SOC/CRAC binding polypeptidewith a sample that is believed to contain one or more SOC/CRACmolecules, under conditions to form a complex of the SOC/CRAC nucleicacid or the SOC/CRAC binding polypeptide and the SOC/CRAC molecule;

b) detecting the presence of the complex;

c) isolating the SOC/CRAC molecule from the complex; and

d) determining whether the isolated SOC/CRAC molecule has SOC/CRACcalcium channel activity. As used herein “SOC/CRAC calcium channelactivity” refers to the transport of Ca²⁺ into and out of intracellularstores that is mediated by a SOC/CRAC polypeptide. In general, theSOC/CRAC calcium channel activity is initiated by a reduction ordepletion of intracellular calcium stores.

In certain embodiments, the SOC/CRAC nucleic acid is a SOC-2/CRAC-1nucleic acid (e.g., a nucleic acid having SEQ ID NO:27, or complementsthereof); in certain other embodiments, the SOC/CRAC nucleic acid is aSOC-3/CRAC-2 nucleic acid (e.g., a nucleic acid having SEQ ID NO:29, orcomplements thereof); in further embodiments, the SOC/CRAC nucleic acidis a SOC-4/CRAC-3 nucleic acid (e.g., a nucleic acid having SEQ IDNO:31, or complements thereof). In yet other embodiments, the SOC/CRACpolypeptide is a SOC-2/CRAC-1 binding polypeptide (e.g., an antibodythat selectively binds to a SOC-2/CRAC-1 polypeptide). In yet furtherembodiments, the SOC/CRAC polypeptide is a SOC-3/CRAC-2 bindingpolypeptide (e.g., an antibody that selectively binds to a SOC-3/CRAC-2polypeptide). In some embodiments, the SOC/CRAC polypeptide is aSOC-4/CRAC-3 binding polypeptide (e.g., an antibody that selectivelybinds to a SOC-4/CRAC-3 polypeptide). In the preferred embodiments, theisolated binding polypeptides include antibodies and fragments ofantibodies (e.g., Fab, F(ab)₂, Fd and antibody fragments which include aCDR3 region which binds selectively to a SOC-2/CRAC-1, to aSOC-3/CRAC-2, and/or to a SOC-4/CRAC-3 polypeptide). Preferably theisolated binding polypeptides or other binding agents selectively bindto a single SOC/CRAC molecule, i.e., are capable of distinguishingbetween different members of the SOC/CRAC family. Accordingly, one ormore SOC/CRAC binding agents can be contained in a single composition(e.g., a pharmaceutical composition) to identify multiple SOC/CRACmolecules in vivo or in vitro.

According to yet another aspect of the invention, a method foridentifying agents useful in the modulation of SOC/CRAC calcium channelactivity is provided. The method involves:

a) contacting a SOC/CRAC polypeptide with a candidate agent suspected ofmodulating SOC/CRAC calcium channel activity, under conditionssufficient to allow the candidate agent to interact selectively with(e.g. bind to) the SOC/CRAC polypeptide;

b) detecting a Ca²⁺ concentration of step (b) associated with theSOC/CRAC calcium channel activity of the SOC/CRAC polypeptide in thepresence of the candidate agent; and

c) comparing the Ca²⁺ concentration of step (b) with a control Ca²⁺concentration of a SOC/CRAC polypeptide in the absence of the candidateagent to determine whether the candidate agent modulates (increases ordecreases) SOC/CRAC calcium channel activity.

According to another aspect of the invention, a method for identifyingagents useful in the modulation of a SOC/CRAC polypeptide kinaseactivity is provided. The method involves:

a) contacting a SOC/CRAC polypeptide with kinase activity with acandidate agent suspected of modulating SOC/CRAC kinase activity, underconditions sufficient to allow the candidate agent to interact with theSOC/CRAC polypeptide and modulate its kinase activity;

b) detecting a kinase activity associated with the SOC/CRAC polypeptidein the presence of the candidate agent; and

c) comparing the kinase activity of step (b) with a control kinaseactivity of a SOC/CRAC polypeptide in the absence of the candidate agentto determine whether the candidate agent modulates (increases ordecreases) SOC/CRAC kinase activity. In some embodiments the SOC/CRACpolypeptide comprises amino acids 999–1180 of the SOC-2/CRAC-1polypeptide (SEQ ID NO:24), or a fragment thereof that retains thekinase activity.

According to yet another aspect of the invention, a method fordetermining the level of expression of a SOC/CRAC polypeptide in asubject is provided. The method involves:

a) measuring the expression of a SOC/CRAC polypeptide in a test sample,and

b) comparing the measured expression of the SOC/CRAC polypeptide in thetest sample to the expression of a SOC/CRAC polypeptide in a controlcontaining a known level of expression to determine the level ofSOC/CRAC expression in the subject. Expression is defined as SOC/CRACmRNA expression or SOC/CRAC polypeptide expression. Various methods canbe used to measure expression. The preferred embodiments of theinvention utilize PCR and Northern blotting for measuring mRNAexpression, and monoclonal or polyclonal SOC/CRAC antisera as reagentsfor measuring SOC/CRAC polypeptide expression. In preferred embodiments,the SOC/CRAC molecule (nucleic acid and/or polypeptide) is SOC-2/CRAC-1.In other preferred embodiments, the SOC/CRAC molecule is SOC-3/CRAC-2.In yet further preferred embodiments, the SOC/CRAC molecule isSOC-4/CRAC-3. In certain embodiments, the test samples include biopsysamples and biological fluids such as blood. The method is useful, e.g.,for assessing the presence or absence or stage of a proliferativedisorder in a subject.

The invention also contemplates kits comprising a package includingassays for SOC/CRAC epitopes, SOC/CRAC nucleic acids, and instructions,and optionally related materials such as controls, for example, anumber, color chart, or an epitope of the expression product of theforegoing isolated nucleic acid molecules of the invention forcomparing, for example, the level of SOC/CRAC polypeptides or SOC/CRACnucleic acid forms (wild-type or mutant) in a test sample to the levelin a control sample having a known amount of a SOC/CRAC nucleic acid orSOC/CRAC polypeptide. This comparison can be used to assess in a subjecta risk of developing a cancer or the progression of a cancer. The kitsmay also include assays for other known genes, and expression productsthereof, associated with, for example, proliferative disorders (e.g.,BRCA, p53, etc.). In a preferred embodiment, the kit comprises a packagecontaining: (a) a binding agent that selectively binds to an isolatednucleic acid of the invention or an expression product thereof to obtaina measured test value, (b) a control containing a known amount of aSOC/CRAC nucleic acid or a SOC/CRAC polypeptide to obtain a measuredcontrol value, and (c) instructions for comparing the measured testvalue to the measured control value to determine the amount of SOC/CRACnucleic acid or expression product thereof in a sample.

The invention provides isolated nucleic acid molecules, unique fragmentsthereof, expression vectors containing the foregoing, and host cellscontaining the foregoing. The invention also provides isolated bindingpolypeptides and binding agents which bind such polypeptides, includingantibodies, and pharmaceutical compositions containing any of thecompositions of the invention. The foregoing can be used, inter alia, inthe diagnosis or treatment of conditions characterized by the aberrantexpression levels and/or the presence of mutant forms of a SOC/CRACnucleic acid or polypeptide. The invention also provides methods foridentifying agents that alter the function of the SOC/CRAC polypeptide.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a partial nucleotide sequence of the human SOC-2/CRAC-1cDNA.

SEQ ID NO:2 is the predicted amino acid sequence of the translationproduct of human SOC-2/CRAC-1 cDNA (SEQ ID NO:1).

SEQ ID NO:3 is a partial nucleotide sequence of the human SOC-2/CRAC-1cDNA.

SEQ ID NO:4 is the predicted amino acid sequence of the translationproduct of human SOC-2/CRAC-1 cDNA (SEQ ID NO:3).

SEQ ID NO:5 is a partial nucleotide sequence of the human SOC-2/CRAC-1cDNA.

SEQ ID NO:6 is the predicted amino acid sequence of the translationproduct of human SOC-2/CRAC-1 cDNA (SEQ ID NO:5).

SEQ ID NO:7 is a partial nucleotide sequence of the mouse homologue(mSOC-2/CRAC-1) of the human SOC-2/CRAC-1 cDNA.

SEQ ID NO:8 is the predicted amino acid sequence of the translationproduct of the mSOC-2/CRAC-1 cDNA (SEQ ID NO:7).

SEQ ID NO:9 is the nucleotide sequence of the mouse MLSN-1 (SOC-1) cDNA.

SEQ ID NO:10 is the predicted amino acid sequence of the translationproduct of the mouse MLSN-1 (SOC-1) cDNA (SEQ ID NO:9).

SEQ ID NO:11 is the nucleotide sequence of a human calcium channel cDNAwith GenBank Acc. no.: AB001535.

SEQ ID NO:12 is the predicted amino acid sequence of the translationproduct of the human calcium channel cDNA with GenBank Acc. no.:AB001535 (SEQ ID NO:11).

SEQ ID NO:13 is the amino acid sequence of a C. Elegans polypeptide atthe c05c12.3 locus.

SEQ ID NO:14 is the amino acid sequence of a C. Elegans polypeptide atthe F54D1 locus.

SEQ ID NO:15 is the amino acid sequence of a C. Elegans polypeptide atthe t01H8 locus.

SEQ ID NO:16 is the nucleotide sequence of a mouse kidney cDNA withGenBank Acc. no.: A1226731.

SEQ ID NO:17 is the predicted amino acid sequence of the translationproduct of the mouse kidney cDNA with GenBank Acc. no.: AI226731 (SEQ IDNO:16).

SEQ ID NO:18 is the nucleotide sequence of a human brain cDNA withGenBank Acc. no.: H18835.

SEQ ID NO:19 is the predicted amino acid sequence of the translationproduct of the human brain cDNA with GenBank Acc. no.: H18835 (SEQ IDNO:18).

SEQ ID NO:20 is the nucleotide sequence of the human EST with GenBankAcc. no.: AA419592.

SEQ ID NO:21 is the nucleotide sequence of the human EST with GenBankAcc. no.: AA419407.

SEQ ID NO:22 is the nucleotide sequence of the mouse EST with GenBankAcc. no.: AI098310.

SEQ ID NO:23 is a partial nucleotide sequence of the human SOC-2/CRAC-1cDNA that contains the SOC-2/CRAC-1 sequences of SEQ ID NO:1, SEQ IDNO:3, and SEQ ID NO:5.

SEQ ID NO:24 is the predicted amino acid sequence of the translationproduct of human SOC-2/CRAC-1 cDNA (SEQ ID NO:23).

SEQ ID NO:25 is a partial nucleotide sequence of the human SOC-3/CRAC-2cDNA.

SEQ ID NO:26 is the predicted amino acid sequence of the translationproduct of human SOC-3/CRAC-2 cDNA (SEQ ID NO:25).

SEQ ID NO:27 is the full nucleotide sequence of the human SOC-2/CRAC-1cDNA.

SEQ ID NO:28 is the predicted amino acid sequence of the translationproduct of human SOC-2/CRAC-1 cDNA (SEQ ID NO:27).

SEQ ID NO:29 is the full nucleotide sequence of the human SOC-3/CRAC-2cDNA.

SEQ ID NO:30 is the predicted amino acid sequence of the translationproduct of human SOC-3/CRAC-2 cDNA (SEQ ID NO:29).

SEQ ID NO:31 is the full nucleotide sequence of the human SOC-4/CRAC-3cDNA.

SEQ ID NO:32 is the predicted amino acid sequence of the translationproduct of human SOC-4/CRAC-3 cDNA (SEQ ID NO:31).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the intron/exon organization of thechicken SOC-2/CRAC-1 genomic sequence, as well as the putativetransmembrane (TM) domains, and the targeting constructs utilized in theknockout experiments.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention involves the partial cloning of cDNAsencoding members of a novel family of calcium channel polypeptides,referred to herein as “SOC/CRAC” (designated “SOC” or “CRAC” or “ICRAC”,for Store Operated Channels or Calcium Release Activated Channels, orCECH). Although not intending to be bound to any particular mechanism ortheory, we believe that a SOC/CRAC family member is a transmembranecalcium channel that modulates Ca²⁺ flux “into” and “out of” a cell; incertain instances it may be activated upon depletion of Ca²⁺ fromintracellular calcium stores, allowing Ca²⁺ influx into the cell.

The first three isolated SOC/CRAC members disclosed herein, define a newfamily of calcium channels which is distinct from previously describedcalcium channels, such as voltage gated calcium channels, ryanodinereceptor/inositol-1,4,5-triphosphate receptor channels, and TransientReceptor Potential (TRP) channels. The SOC/CRAC family of calciumchannels exhibits high selectivity (with a P_(Ca)/P_(Na) ratio near1000), a unitary conductance below the detection level of the patchclamp method (the conductance estimated at approximately 0.2picosiemens), and are subject to inhibition by high intracellularcalcium levels. Although not intending to be bound to any particularmechanism or theory, we believe that SOC/CRAC calcium channels areresponsible for the majority of, for example, calcium entry which occurswhen intracellular calcium stores are depleted, and that SOC/CRACcurrents are important for initiating various types of calcium-dependentprocesses. Thus, we believe that SOC/CRAC calcium channels play animportant role in cellular calcium homeostasis by, e.g., modulating thesupply of calcium to refill intracellular stores when depleted.

The isolated full-length sequence of a representative, first member ofthe SOC/CRAC family, human SOC/CRAC nucleic acid (cDNA), SOC-2/CRAC-1,is represented as the nucleic acid of SEQ ID NO:27. This nucleic acidsequence codes for the SOC-2/CRAC-1 polypeptide with the predicted aminoacid sequence disclosed herein as SEQ ID NO:28. A. homologous mouse cDNAsequence (>90% identity to the human at the nucleotide level) isrepresented as the nucleic acid of SEQ ID NO:7, and codes for a uniquefragment of a mouse SOC-2/CRAC-1 polypeptide having the predicted,partial amino acid sequence represented as SEQ ID NO:8. Analysis of theSOC-2/CRAC-1 partial sequence by comparison to nucleic acid and proteindatabases show that SOC-2/CRAC-1 shares a limited homology to mouseMLSN-1 (SOC-1, SEQ ID NOs: 9 and 10). Limited homology is also sharedbetween SOC-2/CRAC-1 and three C. Elegans polypeptides (SEQ ID NOs:13,14, and 15). We further believe that SOC-2/CRAC-1 plays a role in theregulation of cellular Ca²⁺ fluxing and, in particular, lymphocyte Ca²⁺fluxing.

A second member of the human SOC/CRAC family of calcium channels,SOC-3/CRAC-2, is represented as the nucleic acid of SEQ ID NO:29, andcodes for the human SOC-3/CRAC-2 polypeptide having the predicted aminoacid sequence represented as SEQ ID NO:30 (this molecule may also bereferred to as CECH2). SOC-3/CRAC-2 is predominantly expressed in humanhematopoietic cells (including peripheral blood lymphocytes, liver, bonemarrow, spleen, thymus, lymph nodes, heart, and kidney. Expression canalso be detected (at lesser levels) in brain, skeletal muscle colon,small intestine, placenta, lung, and cells (cell lines) such as HL-60,HeLa, K562, MOLT-4, SW480, A459, and G361.

A third member of the human SOC/CRAC family of calcium channels,SOC-4/CRAC-3, is represented as the nucleic acid of SEQ ID NO:31, andcodes for the human SOC-4/CRAC-3 polypeptide having the predicted aminoacid sequence represented as SEQ ID NO:32 (this molecule may also bereferred to as CECH6). It specifically expressed in the prostategland/cells.

As used herein, a SOC/CRAC calcium channel nucleic acid (also referredto herein as a “SOC/CRAC nucleic acid” refers to a nucleic acid moleculewhich: (1) hybridizes under stringent conditions to one or more of thenucleic acids having the sequences of SEQ ID NOs 7, 27, 29, and/or 31(sequences of the mouse and human SOC-2/CRAC-1, human SOC-3/CRAC-2, andhuman SOC-4/CRAC-3 nucleic acids), and (2) codes for a SOC-2/CRAC-1, aSOC-3/CRAC-2 or a SOC-4/CRAC-3 calcium channel polypeptide,respectively, or unique fragments of said SOC-2/CRAC-1, SOC-3/CRAC-2, orSOC-4/CRAC-3 polypeptide.

As used herein, a SOC/CRAC calcium channel polypeptide (also referred toherein as a “SOC/CRAC polypeptide”) refers to a polypeptide that iscoded for by a SOC-2/CRAC-1, a SOC-3/CRAC-2, and/or a SOC-4/CRAC-3nucleic acid. Preferably, the above-identified SOC/CRAC polypeptidesmediate transport of calcium into and out of a cell.

SOC/CRAC polypeptides also are useful as immunogenic molecules for thegeneration of binding polypeptides (e.g., antibodies) which bindselectively to SOC/CRAC (e.g., SOC-2/CRAC-1, SOC-3/CRAC-2, and/orSOC-4/CRAC-3) polypeptides. Such antibodies can be used in diagnosticassays to identify and/or quantify the presence of a SOC/CRACpolypeptide in a sample, such as a biological fluid or biopsy sample.SOC/CRAC polypeptides further embrace functionally equivalent fragments,variants, and analogs of the preferred SOC/CRAC polypeptides, providedthat the fragments, variants, and analogs also are useful in mediatingcalcium transport into and out of intracellular calcium stores.

As used herein, “SOC/CRAC calcium channel activity” refers to Ca²⁺transport (“Ca²⁺ fluxing”) across the plasma membrane that is mediatedby a SOC/CRAC calcium channel polypeptide. The SOC/CRAC calcium channelpolypeptide typically has one or more of the following properties: highselectivity, a unitary conductance below the detection level of thepatch clamp method, and are subject to inhibition by high intracellularcalcium levels. Such activity can be easily detected using standardmethodology well known in the art. See, e.g., the Examples and Neher,E., “Ion channels for communication between and within cells”, Science,1992; 256:498–502; and Hoth, M., and Penner, R., “Depletion ofintracellular calcium stores activates a calcium current in mast cells”,Nature, 1992; 355 (6358):353–6.

According to one aspect of the invention, isolated nucleic acidmolecules which code for one or more member(s) of the SOC/CRAC family ofcalcium channel polypeptides are provided. The isolated nucleic acidmolecules are selected from the following groups:

(a) nucleic acid molecules which hybridize under stringent conditions toone or more nucleic acid molecules selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and which code fora SOC/CRAC polypeptide;

(b) deletions, additions and substitutions of (a) which code for arespective SOC/CRAC polypeptide;

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b) or (c).

In certain embodiments, the isolated nucleic acid molecule comprises oneor more of nucleotides 1–1212 of SEQ ID NO:1; nucleotides 1–739 of SEQID NO:3; nucleotides 1–1579 of SEQ ID NO:5; nucleotides 1–5117 of SEQ IDNO:23; the mouse homolog for SOC-2/CRAC-1 corresponding to SEQ ID NO:7;nucleotides 1–2180 of SEQ ID NO:25; nucleotides 382–5976 of SEQ IDNO:27; nucleotides 73–3714 of SEQ ID NO:29; and nucleotides 23–3434 ofSEQ ID NO:31. In yet other embodiments, the isolated nucleic acidmolecule comprises a molecule which encodes a polypeptide having one ormore sequences selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and SEQ ID NO:32.

According to yet another aspect of the invention, an isolated nucleicacid molecule is provided which is selected from the group consistingof:

(a) a unique fragment of a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, (ofsufficient length to represent a sequence unique within the humangenome); and (b) complements of (a), provided that the unique fragmentincludes a sequence of contiguous nucleotides which is not identical toa sequence in the prior art as represented by the sequence groupconsisting of: (1) sequences having the SEQ ID NOs or GenBank accessionnumbers of Table I, (2) complements of (1), and (3) fragments of (1) and(2).

In some embodiments, the sequence of contiguous nucleotides is selectedfrom the group consisting of (1) at least two contiguous nucleotidesnonidentical to the sequence group, (2) at least three contiguousnucleotides nonidentical to the sequence group, (3) at least fourcontiguous nucleotides nonidentical to the sequence group, (4) at leastfive contiguous nucleotides nonidentical to the sequence group, (5) atleast six contiguous nucleotides nonidentical to the sequence group, (6)at least seven contiguous nucleotides nonidentical to the sequencegroup.

In other embodiments, the unique fragment has a size selected from thegroup consisting of at least: 8 nucleotides, 10 nucleotides, 12nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20,nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides andevery integer length therebetween.

According to another aspect of the invention, expression vectors andhost cells containing (e.g., transformed or transfected with) expressionvectors comprising the nucleic acid molecules disclosed herein operablylinked to a promoter are provided. In certain preferred embodiments, thehost cells are eukaryotic cells.

The isolated nucleic acid molecules disclosed herein have variousutilities, including their use as probes and primers to identifyadditional members of the SOC/CRAC family of calcium channels, asdiagnostic reagents for identifying the presence of SOC/CRACpolypeptides in biological or other samples, and as agents forgenerating SOC/CRAC binding polypeptides (e.g., antibodies) that can beused as reagents in diagnostic and therapeutic assays to identify thepresence, absence, and/or amounts of a SOC/CRAC nucleic acid orpolypeptide in a biological or other sample.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readilymanipulatable by recombinant DNA techniques well known in the art. Thus,a nucleotide sequence contained in a vector in which 5′ and 3′restriction sites are known or for which polymerase chain reaction (PCR)primer sequences have been disclosed is considered isolated but anucleic acid sequence existing in its native state in its natural hostis not. An isolated nucleic acid may be substantially purified, but neednot be. For example, a nucleic acid that is isolated within a cloning orexpression vector is not pure in that it may comprise only a tinypercentage of the material in the cell in which it resides. Such anucleic acid is isolated, however, as the term is used herein because itis readily manipulatable by standard techniques known to those ofordinary skill in the art.

As used herein with respect to polypeptides (discussed below), the term“isolated” means separated from its native environment in sufficientlypure form so that it can be manipulated or used for any one of thepurposes of the invention. Thus, isolated means sufficiently pure to beused (i) to raise and/or isolate antibodies, (ii) as a reagent in anassay, or (iii) for sequencing, etc.

Homologs and alleles of the SOC/CRAC nucleic acids of the invention canbe identified by conventional techniques. Thus, an aspect of theinvention is those nucleic acid sequences which code for SOC/CRACpolypeptides and which hybridize to a nucleic acid molecules selectedfrom a group consisting of the nucleic acid of SEQ ID NO: 1, the nucleicacid of SEQ ID NO:3, the nucleic acid of SEQ ID NO:5, the nucleic acidof SEQ ID NO:7, the nucleic acid of SEQ ID NO:23, the nucleic acid ofSEQ ID NO:25, the nucleic acid of SEQ ID NO:27, the nucleic acid of SEQID NO:29, and the nucleic acid of SEQ ID NO:31, under stringentconditions. The term “stringent conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references which compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. More specifically,stringent conditions, as used herein, refers, for example, tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5mMNaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodiumchloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; andEDTA is ethylenediaminetetracetic acid. After hybridization, themembrane upon which the DNA is transferred is washed at 2×SSC at roomtemperature and then at 0.1×SSC/0.1×SDS at temperatures up to 68° C.

There are other conditions, reagents, and so forth which can be used,and would result in a similar degree of stringency. The skilled artisanwill be familiar with such conditions, and thus they are not given here.It will be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of the SOC/CRAC nucleic acids of the invention.The skilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

In general homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, and/or SEQ ID NO:31, and SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and/or SEQ ID NO:32, respectively. In someinstances sequences will share at least 50% nucleotide identity and/orat least 65% amino acid identity and in still other instances sequenceswill share at least 60% nucleotide identity and/or at least 75% aminoacid identity. The homology can be calculated using various, publiclyavailable software tools developed by NCBI (Bethesda, Md.) that can beobtained through the internet (ncbi.nlm.nih.gov/pub/). Exemplary toolsinclude the BLAST system available at ncbi.nlm.nih.gov. Pairwise andClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVector sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acids also are embraced by the invention.

In screening for SOC/CRAC related genes, such as homologs and alleles ofSOC-2/CRAC-1 and/or SOC-3/CRAC-2, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film or a phosphoimager plate todetect the radioactive signal.

Given that the expression of the SOC/CRAC gene is prominent in certainhuman tissues (e.g., SOC-2/CRAC-1: lymphoid tissue/heart, SOC-3/CRAC-2:kidney/colon, SOC-4/CRAC-3: prostate), and given the teachings herein ofpartial human SOC/CRAC cDNA clones, full-length and other mammaliansequences corresponding to the human SOC/CRAC partial nucleic acidsequences can be isolated from, for example, a cDNA library preparedfrom one or more of the tissues in which SOC-2/CRAC-1 expression isprominent, SOC-3/CRAC-2 is prominent, and/or SOC-4/CRAC-3 expression isprominent, using standard colony hybridization techniques.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating SOC/CRACpolypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

The invention also provides isolated unique fragments of an isolatednucleic acid molecule selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, and SEQ ID NO:31. A unique fragment is one thatis a ‘signature’ for the larger nucleic acid. For example, the uniquefragment is long enough to assure that its precise sequence is not foundin molecules within the human genome outside of the SOC/CRAC nucleicacids defined above (and human alleles). Those of ordinary skill in theart may apply no more than routine procedures to determine if a fragmentis unique within the human genome.

Unique fragments, however, exclude fragments completely composed of thenucleotide sequences of any of GenBank accession numbers and SEQ ID NOslisted in Table I (SEQ ID NO:9, AB001535, A1226731, H18835, AA419592,AA261842, AA419407, A1098310, AA592910, D86107, AF071787, Z77132,Z83117, Z68333, AA708532, AA551759, AA932133, R47363, N31660, AC005538,AA654650, AA370110, AA313170, AA493512, A1670079, A1671853, AC005538,AA654650, AA370110, AA313170, AA493512, AI670079, AI671853), or otherpreviously published sequences as of the filing date of thisapplication.

A fragment which is completely composed of the sequence described in theforegoing GenBank deposits and SEQ ID NO:9, is one which does notinclude any of the nucleotides unique to the sequences of the invention.Thus, a unique fragment must contain a nucleotide sequence other thanthe exact sequence of those in GenBank or fragments thereof. Thedifference may be an addition, deletion or substitution with respect tothe GenBank sequence or it may be a sequence wholly separate from theGenBank sequence.

Unique fragments can be used as probes in Southern and Northern blotassays to identify such nucleic acids, or can be used in amplificationassays such as those employing PCR. As known to those skilled in theart, large probes such as 200, 250, 300 or more nucleotides arepreferred for certain uses such as Southern and Northern blots, whilesmaller fragments will be preferred for uses such as PCR. Uniquefragments also can be used to produce fusion proteins for generatingantibodies or determining binding of the polypeptide fragments, asdemonstrated in the Examples, or for generating immunoassay components.Likewise, unique fragments can be employed to produce nonfused fragmentsof the SOC/CRAC polypeptides, useful, for example, in the preparation ofantibodies, immunoassays or therapeutic applications. Unique fragmentsfurther can be used as antisense molecules to inhibit the expression ofSOC/CRAC nucleic acids and polypeptides, respectively.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ IDNO:31, and complements thereof, will require longer segments to beunique while others will require only short segments, typically between12 and 32 nucleotides long (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases) or more, up to theentire length of the disclosed sequence. As mentioned above, thisdisclosure intends to embrace each and every fragment of each sequence,beginning at the first nucleotide, the second nucleotide and so on, upto 8 nucleotides short of the end, and ending anywhere from nucleotidenumber 8, 9, 10 and so on for each sequence, up to the very lastnucleotide, (provided the sequence is unique as described above).Virtually any segment of the region of SEQ ID NO:1 beginning atnucleotide 1 and ending at nucleotide 1212, or SEQ ID NO:3 beginning atnucleotide 1 and ending at nucleotide 739, or SEQ ID NO:5 beginning atnucleotide 1 and ending at nucleotide 1579, or SEQ ID NO:7 beginning atnucleotide 1 and ending at nucleotide 3532, or SEQ ID NO:23 beginning atnucleotide 1 and ending at nucleotide 5117, SEQ ID NO:25 beginning atnucleotide 1 and ending at nucleotide 2180, SEQ ID NO:27 beginning atnucleotide 1 and ending at nucleotide 7419, or SEQ ID NO:29 beginning atnucleotide 1 and ending at nucleotide 4061, or SEQ ID NO:31 beginning atnucleotide 1 and ending at nucleotide 4646, or complements thereof, thatis 20 or more nucleotides in length will be unique. Those skilled in theart are well versed in methods for selecting such sequences, typicallyon the basis of the ability of the unique fragment to selectivelydistinguish the sequence of interest from other sequences in the humangenome of the fragment to those on known databases typically is all thatis necessary, although in vitro confirmatory hybridization andsequencing analysis may be performed.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a SOC/CRACpolypeptide, to decrease SOC/CRAC calcium channel activity. When usingantisense preparations of the invention, slow intravenous administrationis preferred.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ IDNO:31, or upon allelic or homologous genomic and/or cDNA sequences, oneof skill in the art can easily choose and synthesize any of a number ofappropriate antisense molecules for use in accordance with the presentinvention. In order to be sufficiently selective and potent forinhibition, such antisense oligonucleotides should comprise at least 10and, more preferably, at least 15 consecutive bases which arecomplementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., Nat. Med.1(11):1116–1118, 1995). Most preferably, the antisense oligonucleotidescomprise a complementary sequence of 20–30 bases. Althougholigonucleotides may be chosen which are antisense to any region of thegene or mRNA transcripts, in preferred embodiments the antisenseoligonucleotides correspond to N-terminal or 5′ upstream sites such astranslation initiation, transcription initiation or promoter sites. Inaddition, 3′-untranslated regions may be targeted by antisenseoligonucleotides. Targeting to mRNA splicing sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In addition, the antisense is targeted, preferably, to sites in whichmRNA secondary structure is not expected (see, e.g., Sainio et al., CellMol. Neurobiol. 14(5):439–457, 1994) and at which proteins are notexpected to bind. Finally, although, SEQ ID No:1 discloses a cDNAsequence, one of ordinary skill in the art may easily derive the genomicDNA corresponding to this sequence. Thus, the present invention alsoprovides for antisense oligonucleotides which are complementary to thegenomic DNA corresponding to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:23, SEQ ID NO:25, in SEQ ID NO:27, SEQ ID NO:29, andSEQ ID NO:31. Similarly, antisense to allelic or homologous SOC/CRACcDNAs and genomic DNAs are enabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding SOC/CRACpolypeptides, together with pharmaceutically acceptable carriers.Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The invention also involves expression vectors coding for SOC/CRACproteins and fragments and variants thereof and host cells containingthose expression vectors. Virtually any cells, prokaryotic oreukaryotic, which can be transformed with heterologous DNA or RNA andwhich can be grown or maintained in culture, may be used in the practiceof the invention. Examples include bacterial cells such as E. coli andeukaryotic cells such as mouse, hamster, pig, goat, primate, yeast,xenopous, etc. They may be of a wide variety of tissue types, includingmast cells, fibroblasts, oocytes and lymphocytes, and they may beprimary cells or cell lines. Specific examples include CHO cells and COScells. Cell-free transcription systems also may be used in lieu ofcells.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

According to yet another aspect of the invention, isolated SOC/CRACpolypeptides are provided. Preferably, the isolated SOC/CRACpolypeptides are encoded by the isolated SOC/CRAC nucleic acid moleculesdisclosed herein. More preferably, the isolated SOC/CRAC polypeptides ofthe invention are encoded by the nucleic acid molecules having SEQ IDNos. 1, 3, 5, 7, 23, 25, 27, 29, and 31. In yet other embodiments, theisolated SOC/CRAC polypeptides of the invention have an amino acidsequence selected from the group consisting of SEQ ID Nos. 2, 4, 6, 8,24, 26, 28, 30 and 32. Preferably, the isolated SOC/CRAC polypeptidesare of sufficient length to represent a sequence unique within the humangenome. Thus, the preferred embodiments include a sequence of contiguousamino acids which is not identical to a prior art sequence asrepresented by the sequence group consisting of the contiguous aminoacids identified in Table II (SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and GenBank Acc. Nos. AB001535,AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759,AA932133, R47363, N31660, NP003298, CAB00861, NP002411, CAA92726,CAB05572).

In certain embodiments, the isolated SOC/CRAC polypeptides areimmunogenic and can be used to generate binding polypeptides (e.g.,antibodies) for use in diagnostic and therapeutic applications. Suchbinding polypeptides also are useful for detecting the presence,absence, and/or amounts of a SOC/CRAC nucleic acid or polypeptide in asample such as a biological fluid or biopsy sample. Preferably, theSOC/CRAC polypeptides that are useful for generating bindingpolypeptides are unique polypeptides and, therefore, binding of theantibody to a SOC/CRAC polypeptide in a sample is selective for theSOC/CRAC polypeptide.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition Cold Spring Harbor Laboratory Press, 1989. Cells are geneticallyengineered by the introduction into the cells of heterologous DNA (RNA)encoding a SOC/CRAC polypeptide or fragment or variant thereof. Theheterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710–4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626–630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303–310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

It will also be recognized that the invention embraces the use of theabove described, SOC/CRAC cDNA sequence containing expression vectors,to transfect host cells and cell lines, by these prokaryotic (e.g., E.coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expressionsystems and recombinant baculovirus expression in insect cells).Especially useful are mammalian cells such as mouse, hamster, pig, goat,primate, etc. They may be of a wide variety of tissue types, and includeprimary cells and cell lines. Specific examples include dendritic cells,U293 cells, peripheral blood leukocytes, bone marrow stem cells andembryonic stem cells. The invention also permits the construction ofSOC/CRAC gene “knock-outs” in cells and in animals, providing materialsfor studying certain aspects of SOC/CRAC calcium channel activity.

The invention also provides isolated polypeptides (including wholeproteins and partial proteins), encoded by the foregoing SOC/CRACnucleic acids, and include the polypeptides of SEQ ID NO:2, 4, 6, 8, 24,26, 28, 30, 32, and unique fragments thereof. Such polypeptides areuseful, for example, to regulate calcium transport-mediated cell growth,differentiation and proliferation, to generate antibodies, as componentsof immunoassays, etc. Polypeptides can be isolated from biologicalsamples including tissue or cell homogenates, and can also be expressedrecombinantly in a variety of prokaryotic and eukaryotic expressionsystems by constructing an expression vector appropriate to theexpression system, introducing the expression vector into the expressionsystem, and isolating the recombinantly expressed protein. Shortpolypeptides, including antigenic peptides (such as are presented by MHCmolecules on the surface of a cell for immune recognition) also can besynthesized chemically using well-established methods of peptidesynthesis.

A unique fragment of a SOC/CRAC polypeptide, in general, has thefeatures and characteristics of unique fragments as discussed above inconnection with nucleic acids. As will be recognized by those skilled inthe art, the size of the unique fragment will depend upon factors suchas whether the fragment constitutes a portion of a conserved proteindomain. Thus, some regions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/orSEQ ID NO:32, will require longer segments to be unique while otherswill require only short segments, typically between 5 and 12 amino acids(e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long or more, includingeach integer up to the full length, >1,000 amino acids long). Virtuallyany segment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/or SEQ ID NO:32,excluding the ones that share identity with it (the polypeptidesidentified in Table II—SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, and GenBank Acc. Nos. AB001535,AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759,AA932133, R47363, N31660, NP003298, CAB00861, NP002411, CAA92726,CAB05572) that is 9 or more amino acids in length will be unique.

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude Ca²⁺ fluxing, high selectivity, a unitary conductance below thedetection level of the patch clamp method, and/or and are subject toinhibition by high intracellular calcium levels.

One important aspect of a unique fragment is its ability to act as asignature for identifying the polypeptide. Optionally, another aspect ofa unique fragment is its ability to provide an immune response in ananimal. Those skilled in the art are well versed in methods forselecting unique amino acid sequences, typically on the basis of theability of the unique fragment to selectively distinguish the sequenceof interest from non-family members. A comparison of the sequence of thefragment to those on known databases typically is all that is necessary.

The invention embraces variants of the SOC/CRAC polypeptides describedabove. As used herein, a “variant” of a SOC/CRAC polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a SOC/CRAC polypeptide. Modifications whichcreate a SOC/CRAC polypeptide variant are typically made to the nucleicacid which encodes the SOC/CRAC polypeptide, and can include deletions,point mutations, truncations, amino acid substitutions and addition ofamino acids or non-amino acid moieties to: 1) reduce or eliminate acalcium channel activity of a SOC/CRAC polypeptide; 2) enhance aproperty of a SOC/CRAC polypeptide, such as protein stability in anexpression system or the stability of protein—protein binding; 3)provide a novel activity or property to a SOC/CRAC polypeptide, such asaddition of an antigenic epitope or addition of a detectable moiety; or4) to provide equivalent or better binding to a SOC/CRAC polypeptidereceptor or other molecule. Alternatively, modifications can be madedirectly to the polypeptide, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as biotin, addition of afatty acid, and the like. Modifications also embrace fusion proteinscomprising all or part of the SOC/CRAC amino acid sequence. One of skillin the art will be familiar with methods for predicting the effect onprotein conformation of a change in protein sequence, and can thus“design” a variant SOC/CRAC polypeptide according to known methods. Oneexample of such a method is described by Dahiyat and Mayo in Science278:82–87, 1997, whereby proteins can be designed de novo. The methodcan be applied to a known protein to vary only a portion of thepolypeptide sequence. By applying the computational methods of Dahiyatand Mayo, specific variants of a SOC/CRAC calcium channel polypeptidecan be proposed and tested to determine whether the variant retains adesired conformation.

Variants can include SOC/CRAC polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aSOC/CRAC polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encodes a SOC/CRAC polypeptidepreferably preserve the amino acid reading frame of the coding sequenceand, preferably, do not create regions in the nucleic acid which arelikely to hybridize to form secondary structures, such as hairpins orloops, which can be deleterious to expression of the variantpolypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant SOC/CRAC polypeptides) which are silentas to the amino acid sequence of the polypeptide, but which providepreferred codons for translation in a particular host. The preferredcodons for translation of a nucleic acid in, e.g., E. coli, are wellknown to those of ordinary skill in the art. Still other mutations canbe made to the noncoding sequences of a SOC/CRAC gene or cDNA clone toenhance expression of the polypeptide.

The skilled artisan will realize that conservative amino acidsubstitutions may be made in SOC/CRAC polypeptides to providefunctionally equivalent variants of the foregoing polypeptides, i.e, thevariants retain the functional capabilities of the SOC/CRACpolypeptides. As used herein, a “conservative amino acid substitution”refers to an amino acid substitution which does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references which compile such methods, e.g.Molecular Cloning. A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. Exemplary functionallyequivalent variants of the SOC/CRAC polypeptides include conservativeamino acid substitutions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/orSEQ ID NO:32. Conservative substitutions of amino acids includesubstitutions made amongst amino acids within the following groups: (a)M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) St T; (f) Q, N; and(g) E, D.

Thus functionally equivalent variants of SOC/CRAC polypeptides, i.e.,variants of SOC/CRAC polypeptides which retain the function of thenatural SOC/CRAC polypeptides, are contemplated by the invention.Conservative amino-acid substitutions in the amino acid sequence ofSOC/CRAC polypeptides to produce functionally equivalent variants ofSOC/CRAC polypeptides typically are made by alteration of a nucleic acidencoding SOC/CRAC polypeptides (e.g., SEQ ID NOs:1, 3, 5, 7, 23, 25, 27,29, 31). Such substitutions can be made by a variety of methods known toone of ordinary skill in the art. For example, amino acid substitutionsmay be made by PCR-directed mutation, site-directed mutagenesisaccording to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A.82: 488–492, 1985), or by chemical synthesis of a gene encoding aSOC/CRAC polypeptide. The activity of functionally equivalent fragmentsof SOC/CRAC polypeptides can be tested by cloning the gene encoding thealtered SOC/CRAC polypeptide into a bacterial or mammalian expressionvector, introducing the vector into an appropriate host cell, expressingthe altered SOC/CRAC polypeptide, and testing for a functionalcapability of the SOC/CRAC polypeptides as disclosed herein (e.g.,SOC/CRAC calcium channel activity).

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof SOC/CRAC polypeptides, including the isolation of the completeSOC/CRAC polypeptide. A variety of methodologies well-known to theskilled practitioner can be utilized to obtain isolated SOC/CRACmolecules. The polypeptide may be purified from cells which naturallyproduce the polypeptide by chromatographic means or immunologicalrecognition. Alternatively, an expression vector may be introduced intocells to cause production of the polypeptide. In another method, mRNAtranscripts may be microinjected or otherwise introduced into cells tocause production of the encoded polypeptide. Translation of SOC/CRACmRNA in cell-free extracts such as the reticulocyte lysate system alsomay be used to produce SOC/CRAC polypeptides. Those skilled in the artalso can readily follow known methods for isolating SOC/CRACpolypeptides. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from SOC/CRAC polypeptides. A dominant negativepolypeptide is an inactive variant of a protein, which, by interactingwith the cellular machinery, displaces an active protein from itsinteraction with the cellular machinery or competes with the activeprotein, thereby reducing the effect of the active protein. For example,a dominant negative receptor which binds a ligand but does not transmita signal in response to binding of the ligand can reduce the biologicaleffect of expression of the ligand. Likewise, a dominant negativeinactive SOC/CRAC calcium channel which interacts normally with the cellmembrane but which does not mediate calcium transport can reduce calciumtransport in a cell. Similarly, a dominant negative transcription factorwhich binds to a promoter site in the control region of a gene but doesnot increase gene transcription can reduce the effect of a normaltranscription factor by occupying promoter binding sites withoutincreasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. See, e.g.,U.S. Pat. No. 5,580,723 and Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press.The skilled artisan then can test the population of mutagenizedpolypeptides for diminution in a selected and/or for retention of suchan activity. Other similar methods for creating and testing dominantnegative variants of a protein will be apparent to one of ordinary skillin the art.

According to another aspect, the invention provides a method forisolating a SOC/CRAC molecule having SOC/CRAC calcium channel activity.The method involves contacting a binding molecule that is a SOC/CRACnucleic acid or a SOC/CRAC binding polypeptide with a sample containingone or more SOC/CRAC molecules under conditions that allow such binding(see earlier discussion) to form a complex, detecting the presence ofthe complex, isolating the SOC/CRAC molecule from the complex, anddetermining whether the isolated SOC/CRAC molecule has SOC/CRAC calciumchannel activity. Thus, the invention is useful for identifying andisolating full length complementary (cDNA) or genomic nucleic acidsencoding SOC/CRAC polypeptides having SOC/CRAC calcium channel activity.Identification and isolation of such nucleic acids and polypeptides maybe accomplished by hybridizing/binding, under appropriate conditionswell known in the art, libraries and/or restriction enzyme-digestedhuman nucleic acids, with a labeled SOC/CRAC molecular probe. As usedherein, a “label” includes molecules that are incorporated into, forexample, a SOC/CRAC molecule (nucleic acid or peptide), that can bedirectly or indirectly detected. A wide variety of detectable labels arewell known in the art that can be used, and include labels that providedirect detection (e.g., radioactivity, luminescence, optical or electrondensity, etc), or indirect detection (e.g., epitope tag such as the FLAGepitope, enzyme tag such as horseseradish peroxidase, etc.). The labelmay be bound to a SOC/CRAC binding partner, or incorporated into thestructure of the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions,nonradioactive energy transfers, etc. or indirectly detected withantibody conjugates, strepavidin-biotin conjugates, etc. Methods fordetecting the labels are well known in the art. Once a library clone orhybridizing fragment is identified in the hybridization/bindingreaction, it can be further isolated by employing standardisolation/cloning techniques known to those of skill in the art. See,generally, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press. In addition,nucleic acid amplification techniques well known in the art, may also beused to locate splice variants of calcium channel (or calcium channelsubunits) with SOC/CRAC calcium channel activity. Size and sequencedeterminations of the amplification products can reveal splice variants.

The foregoing isolated nucleic acids and polypeptides may then becompared to the nucleic acids and polypeptides of the present inventionin order to identify homogeneity or divergence of the sequences, and befurther characterized functionally to determine whether they belong to afamily of molecules with SOC/CRAC calcium channel activity (formethodology see under the Examples section).

The isolation of the SOC/CRAC cDNA and/or partial sequences thereof alsomakes it possible for the artisan to diagnose a disorder characterizedby an aberrant expression of SOC/CRAC. These methods involve determiningexpression of the SOC/CRAC gene, and/or SOC/CRAC polypeptides derivedtherefrom. In the former situation, such determinations can be carriedout via any standard nucleic acid determination assay, including thepolymerase chain reaction, or assaying with labeled hybridization probesas exemplified below. In the latter situation, such determination can becarried out via any standard immunological assay using, for example,antibodies which bind to the SOC/CRAC protein.

The invention also embraces isolated peptide binding agents which, forexample, can be antibodies or fragments of antibodies (“bindingpolypeptides”), having the ability to selectively bind to SOC/CRACpolypeptides. Antibodies include polyclonal and monoclonal antibodies,prepared according to conventional methodology. In certain embodiments,the invention excludes binding agents (e.g., antibodies) that bind tothe polypeptides encoded by the nucleic acids of SEQ ID NOs:10, 12, 13,14, 15, 17, and 19.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves binding polypeptides of numerous size andtype that bind selectively to SOC/CRAC polypeptides, and complexescontaining SOC/CRAC polypeptides. These binding polypeptides also may bederived also from sources other than antibody technology. For example,such polypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilizedform, as bacterial flagella peptide display libraries or as phagedisplay libraries. Combinatorial libraries also can be synthesized ofpeptides containing one or more amino acids. Libraries further can besynthesized of peptides and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe SOC/CRAC polypeptide or a complex containing a SOC/CRAC polypeptide.This process can be repeated through several cycles of reselection ofphage that bind to the SOC/CRAC polypeptide or complex. Repeated roundslead to enrichment of phage bearing particular sequences. DNA sequenceanalysis can be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds tothe SOC/CRAC polypeptide or complex can be determined. One can repeatthe procedure using a biased library containing inserts containing partor all of the minimal linear portion plus one or more additionaldegenerate residues upstream or downstream thereof. Yeast two-hybridscreening methods also may be used to identify polypeptides that bind tothe SOC/CRAC polypeptides. Thus, the SOC/CRAC polypeptides of theinvention, or a fragment thereof, or complexes of SOC/CRAC can be usedto screen peptide libraries, including phage display libraries, toidentify and select peptide binding polypeptides that selectively bindto the SOC/CRAC polypeptides of the invention. Such molecules can beused, as described, for screening assays, for purification protocols,for interfering directly with the functioning of SOC/CRAC and for otherpurposes that will be apparent to those of ordinary skill in the art.

A SOC/CRAC polypeptide, or a fragment thereof, also can be used toisolate naturally occurring, polypeptide binding partners which mayassociate with the SOC/CRAC polypeptide in the membrane of a cell.Isolation of binding partners may be performed according to well-knownmethods. For example, isolated SOC/CRAC polypeptides can be attached toa substrate, and then a solution suspected of containing an SOC/CRACbinding partner may be applied to the substrate. If the binding partnerfor SOC/CRAC polypeptides is present in the solution, then it will bindto the substrate-bound SOC/CRAC polypeptide. The binding partner thenmay be isolated. Other proteins which are binding partners for SOC/CRAC,may be isolated by similar methods without undue experimentation.

The invention also provides novel kits which could be used to measurethe levels of the nucleic acids of the invention, expression products ofthe invention or anti-SOC/CRAC antibodies. In the case of nucleic aciddetection, pairs of primers for amplifying SOC/CRAC nucleic acids can beincluded. The preferred kits would include controls such as knownamounts of nucleic acid probes, SOC/CRAC epitopes (such as SOC/CRACexpression products) or anti-SOC/CRAC antibodies, as well asinstructions or other printed material. In certain embodiments theprinted material can characterize risk of developing a disorder that ischaracterized by aberrant SOC/CRAC polypeptide expression based upon theoutcome of the assay. The reagents may be packaged in containers and/orcoated on wells in predetermined amounts, and the kits may includestandard materials such as labeled immunological reagents (such aslabeled anti-IgG antibodies) and the like. One kit is a packagedpolystyrene microtiter plate coated with a SOC/CRAC polypeptide and acontainer containing labeled anti-human IgG antibodies. A well of theplate is contacted with, for example, serum, washed and then contactedwith the anti-IgG antibody. The label is then detected. A kit embodyingfeatures of the present invention is comprised of the following majorelements: packaging an agent of the invention, a control agent, andinstructions. Packaging is a box-like structure for holding a vial (ornumber of vials) containing an agent of the invention, a vial (or numberof vials) containing a control agent, and instructions. Individualsskilled in the art can readily modify packaging to suit individualneeds.

Another aspect of the invention is a method for determining the level ofSOC/CRAC expression in a subject. As used herein, a subject is a human,non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. Inall embodiments, human subjects are preferred. Expression is definedeither as SOC/CRAC mRNA expression or SOC/CRAC polypeptide expression.Various methods can be used to measure expression. Preferred embodimentsof the invention include PCR and Northern blotting for measuring mRNAexpression, and monoclonal or polyclonal SOC/CRAC antisera as reagentsto measure SOC/CRAC polypeptide expression. In certain embodiments, testsamples such as biopsy samples, and biological fluids such as blood, areused as test samples. SOC/CRAC expression in a test sample of a subjectis compared to SOC/CRAC expression in control sample to, e.g., assessthe presence or absence or stage of a proliferative disorder (e.g., alymphocyte proliferative disorder) in a subject.

SOC/CRAC polypeptides preferably are produced recombinantly, althoughsuch polypeptides may be isolated from biological extracts.Recombinantly produced SOC/CRAC polypeptides include chimeric proteinscomprising a fusion of a SOC/CRAC protein with another polypeptide,e.g., a polypeptide capable of providing or enhancing protein—proteinbinding, sequence specific nucleic acid binding (such as GAL4),enhancing stability of the SOC/CRAC polypeptide under assay conditions,or providing a detectable moiety, such as green fluorescent protein. Apolypeptide fused to a SOC/CRAC polypeptide or fragment may also providemeans of readily detecting the fusion protein, e.g., by immunologicalrecognition or by fluorescent labeling.

The invention is also useful in the generation of transgenic non-humananimals. As used herein, “transgenic non-human animals” includesnon-human animals having one or more exogenous nucleic acid moleculesincorporated in germ line cells and/or somatic cells. Thus thetransgenic animal include “knockout” animals having a homozygous orheterozygous gene disruption by homologous recombination, animals havingepisomal or chromosomally incorporated expression vectors, etc. Knockoutanimals can be prepared by homologous recombination using embryonic stemcells as is well known in the art. The recombination may be facilitatedusing, for example, the cre/lox system or other recombinase systemsknown to one of ordinary skill in the art. In certain embodiments, therecombinase system itself is expressed conditionally, for example, incertain tissues or cell types, at certain embryonic or post-embryonicdevelopmental stages, inducibly by the addition of a compound whichincreases or decreases expression, and the like. In general, theconditional expression vectors used in such systems use a variety ofpromoters which confer the desired gene expression pattern (e.g.,temporal or spatial). Conditional promoters also can be operably linkedto SOC/CRAC nucleic acid molecules to increase expression of SOC/CRAC ina regulated or conditional manner. Trans-acting negative regulators ofSOC/CRAC calcium channel activity or expression also can be operablylinked to a conditional promoter as described above. Such trans-actingregulators include antisense SOC/CRAC nucleic acids molecules, nucleicacid molecules which encode dominant negative SOC/CRAC molecules,ribozyme molecules specific for SOC/CRAC nucleic acids, and the like.The transgenic non-human animals are useful in experiments directedtoward testing biochemical or physiological effects of diagnostics ortherapeutics for conditions characterized by increased or decreasedSOC/CRAC expression. Other uses will be apparent to one of ordinaryskill in the art.

The invention further provides efficient methods of identifying agentsor lead compounds for agents active at the level of a SOC/CRACpolypeptide (e.g., a SOC/CRAC polypeptide) or SOC/CRAC fragmentdependent cellular function. In particular, such functions includeinteraction with other polypeptides or fragments thereof, and selectivebinding to certain molecules (e.g., agonists and antagonists).Generally, the screening methods involve assaying for compounds whichinterfere with SOC/CRAC calcium channel activity, although compoundswhich enhance SOC/CRAC calcium channel activity also can be assayedusing the screening methods. Such methods are adaptable to automated,high throughput screening of compounds. The target therapeuticindications for pharmacological agents detected by the screening methodsare limited only in that the target cellular function be subject tomodulation by alteration of the formation of a complex comprising aSOC/CRAC polypeptide or fragment thereof and one or more SOC/CRACbinding targets. Target indications include cellular processes modulatedby SOC/CRAC such as Ca²⁺ fluxing, and affected by SOC/CRAC ability toform complexes with other molecules and polypeptides as, for example,may be present in the cell membrane.

A wide variety of assays for pharmacological agents are provided,including, expression assays, labeled in vitro protein—protein bindingassays, electrophoretic mobility shift assays, immunoassays, cell-basedassays such as calcium transport assays, etc. For example, two-hybridscreens are used to rapidly examine the effect of transfected nucleicacids on the intracellular binding of SOC/CRAC or SOC/CRAC fragments tospecific intracellular targets (e.g. a tyrosine kinase). The transfectednucleic acids can encode, for example, combinatorial peptide librariesor cDNA libraries. Convenient reagents for such assays, e.g., GAL4fusion proteins, are known in the art. An exemplary cell-based assayinvolves transfecting a cell with a nucleic acid encoding a SOC/CRACpolypeptide fused to a GAL4 DNA binding domain and a nucleic acidencoding a reporter gene operably linked to a gene expression regulatoryregion, such as one or more GAL4 binding sites. Activation of reportergene transcription occurs when the SOC/CRAC and reporter fusionpolypeptides bind such as to enable transcription of the reporter gene.Agents which modulate a SOC/CRAC polypeptide mediated cell function arethen detected through a change in the expression of reporter gene.Methods for determining changes in the expression of a reporter gene areknown in the art.

In an expression system, for example, a SOC/CRAC polypeptide is attachedto a membrane, the membrane preferably separating two fluid environmentsand being otherwise not permeable to Ca²⁺. Such separation is preferredso that a change in Ca²⁺ concentration on either side of the membrane ismediated only through the attached SOC/CRAC polypeptide. Preferably, aSOC/CRAC polypeptide is expressed in an intact cell and is present onthe cell-membrane (as in physiologic conditions). The cell expressingthe SOC/CRAC polypeptide is preferably a eukaryotic cell, and theSOC/CRAC polypeptide is preferably recombinantly expressed, althoughcells naturally expressing a SOC/CRAC polypeptide may also be used.Synthetic membranes, however, containing SOC/CRAC polypeptides may alsobe used. See, e.g., K. Kiselyov, et al., Functional interaction betweenInsP3 receptors and store-operated Htrp3 channels, Nature 396, 478–82(1998).

The cell expressing the SOC/CRAC polypeptide is incubated underconditions which, in the absence of the candidate agent, permit calciumflux into the cell and allow detection of a reference calciumconcentration. For example, depletion of intracellular calcium storeswith thapsigargin or other agents (Putney, J. W. Jr., in CapacitativeCalcium Entry, R.G. Landes Co. and Chapman & Hall, 1997) would produce agiven level of SOC/CRAC channel activation and a given reference calciumconcentration. Detection of a decrease in the foregoing activities(i.e., a decrease in the intracellular calcium concentration) relativeto the reference calcium concentration indicates that the candidateagent is a lead compound for an agent to inhibit SOC/CRAC calciumchannel activity. Preferred SOC/CRAC polypeptides include thepolypeptides of claim 15.

SOC/CRAC fragments used in the methods, when not produced by atransfected nucleic acid are added to an assay mixture as an isolatedpolypeptide. SOC/CRAC polypeptides preferably are producedrecombinantly, although such polypeptides may be isolated frombiological extracts or chemically synthesized. Recombinantly producedSOC/CRAC polypeptides include chimeric proteins comprising a fusion of aSOC/CRAC protein with another polypeptide, e.g., a polypeptide capableof providing or enhancing protein—protein binding, sequence specificnucleic acid binding (such as GAL4), enhancing stability of the SOC/CRACpolypeptide under assay conditions, or providing a detectable moiety,such as green fluorescent protein or Flag epitope.

The assay mixture is comprised of a SOC/CRAC polypeptide binding target(candidate agent) capable of interacting with a SOC/CRAC polypeptide.While natural SOC/CRAC binding targets may be used, it is frequentlypreferred to use portions (e.g., peptides or nucleic acid fragments) oranalogs (i.e., agents which mimic the SOC/CRAC binding properties of thenatural binding target for purposes of the assay) of the SOC/CRACbinding target so long as the portion or analog provides bindingaffinity and avidity to the SOC/CRAC polypeptide (or fragment thereof)measurable in the assay.

The assay mixture also comprises a candidate agent (binding target,e.g., agonist/antagonist). Typically, a plurality of assay mixtures arerun in parallel with different agent concentrations to obtain adifferent response to the various concentrations. Typically, one ofthese concentrations serves as a negative control, i.e., at zeroconcentration of agent or at a concentration of agent below the limitsof assay detection. Candidate agents encompass numerous chemicalclasses, although typically they are organic compounds. Preferably, thecandidate agents are small organic compounds, i.e., those having amolecular weight of more than 50 yet less than about 2500, preferablyless than about 1000 and, more preferably, less than about 500.Candidate agents comprise functional chemical groups necessary forstructural interactions with polypeptides and/or nucleic acids, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is anucleic acid, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acids as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily modified through conventional chemical, physical, andbiochemical means. Further, known agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents. Non-SOC/CRAC calcium channel agonists and antagonists, forexample, include agents such as dihydropyridines (DHPs),phenylalkylamines, omega conotoxin (omega.-CgTx) andpyrazonoylguanidines.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein—protein, protein-nucleic acid, and/or protein/membrane componentbinding association. Such a reagent may also reduce non-specific orbackground interactions of the reaction components. Other reagents thatimprove the efficiency of the assay such as protease, inhibitors,nuclease inhibitors, antimicrobial agents, and the like may also beused.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidate agent, theSOC/CRAC polypeptide specifically binds the cellular binding target, aportion thereof or analog thereof. The order of addition of components,incubation temperature, time of incubation, and other perimeters of theassay may be readily determined. Such experimentation merely involvesoptimization of the assay parameters, not the fundamental composition ofthe assay. Incubation temperatures typically are between 4° C. and 40°C. Incubation times preferably are minimized to facilitate rapid, highthroughput screening, and typically are between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe SOC/CRAC polypeptide and one or more binding targets is detected byany convenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximum signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of SOC/CRAC polypeptide interactingwith a target molecule typically encodes a directly or indirectlydetectable product, e.g., β-galactosidase activity, luciferase activity,and the like. For cell-free binding assays, one of the componentsusually comprises, or is coupled to, a detectable label. A wide varietyof labels can be used, such as those that provide direct detection(e.g., radioactivity, luminescence, optical or electron density, etc.)or indirect detection (e.g., epitope tag such as the FLAG epitope,enzyme tag such as horseseradish peroxidase, etc.). The label may bebound to a SOC/CRAC binding partner, or incorporated into the structureof the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art.

Of particular importance in any of the foregoing assays and bindingstudies is the use of a specific sequence motif identified in theSOC-2/CRAC-1 polypeptide sequence as a kinase catalytic domain.According to the invention, amino acids 999–1180 of the SOC-2/CRAC-1polypeptide (SEQ ID NO:24) (or a fragment thereof), show a localizedhomology with the catalytic domains of eukaryotic elongation factor-2kinase (eEF-2 kinase, GenBank Acc. no. U93850) and Dictyostelium myocinheavy chain kinase A (MHCK A, GenBank Acc. no. U16856), as disclosed inRyazanov AG, et al., Proc Natl Acad Sci USA, 1997, 94(10):4884–4889.Therefore, according to the invention, a method for identifying agentsuseful in the modulation of SOC/CRAC polypeptide kinase activity isprovided. The method involves contacting a SOC/CRAC polypeptide withkinase activity, that includes, for example, amino acids 999–1180 of theSOC-2/CRAC-1 polypeptide (SEQ ID NO:24) with a candidate agent suspectedof modulating SOC/CRAC kinase activity, under conditions sufficient toallow the candidate agent to interact with the SOC/CRAC polypeptide andmodulate its kinase activity; detecting a kinase activity associatedwith the SOC/CRAC polypeptide in the presence of the candidate agent;and comparing the kinase activity in the previous step with a controlkinase activity of a SOC/CRAC polypeptide in the absence of thecandidate agent to determine whether the candidate agent modulates(increases or decreases) SOC/CRAC kinase activity. Other controls forkinase activity can also be performed at the same time, for example, byutilizing eEF-2 kinase and/or Dictyostelium MHC Kinase A, in a similarmanner to the SOC/CRAC member. Methods for performing such kinaseactivity assays are well known in the art.

The invention thus provides SOC/CRAC-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, SOC/CRAC-specific agentsare useful in a variety of diagnostic and therapeutic applications,especially where disease or disease prognosis is associated with alteredSOC/CRAC and SOC/CRAC calcium channel fluxing characteristics. NovelSOC/CRAC-specific binding agents include SOC/CRAC-specific antibodiesand other natural intracellular and extracellular binding agentsidentified with assays such as two hybrid screens, and non-naturalintracellular and extracellular binding agents identified in screens ofchemical libraries and the like.

In general, the specificity of SOC/CRAC binding to a specific moleculeis determined by binding equilibrium constants. Targets which arecapable of selectively binding a SOC/CRAC polypeptide preferably havebinding equilibrium constants of at least about 107 M⁻¹, more preferablyat least about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. Thewide variety of cell based and cell free assays may be used todemonstrate SOC/CRAC-specific binding. Cell based assays include one,two and three hybrid screens, assays in which SOC/CRAC-mediatedtranscription is inhibited or increased, etc. Cell free assays includeSOC/CRAC-protein binding assays, immunoassays, etc. Other assays usefulfor screening agents which bind SOC/CRAC polypeptides includefluorescence resonance energy transfer (FRET), and electrophoreticmobility shift analysis (EMSA).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a retrovirus, or othervirus; a liposome) can have a targeting molecule attached thereto. Forexample, a molecule such as an antibody specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellcan be bound to or incorporated within the nucleic acid deliveryvehicle. For example, where liposomes are employed to deliver thenucleic acids of the invention, proteins which bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichan agent of the invention is contained in a form within a matrix such asthose described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152,and (b) diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.See, e.g., U.S. Pat. No. 5,670,488, entitled “Adenovirus Vector for GeneTherapy”, issued to Gregory et al., and U.S. Pat. No. 5,672,344,entitled “Viral-Mediated Gene Transfer System”, issued to Kelley et al.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES

As an initial approach to identifying SOC/CRAC channels, we consideredpublicly available data and hypothesized that the followingcharacteristics are likely to be exhibited by SOC/CRAC calcium channels:i) SOC/CRAC calcium channels would be integral membrane proteins related(probably distantly) to one of the known calcium channel families (e.g.voltage gated, ligand gated, Trp), and therefore should have a poreregion formed by a tetramer of 6–7 transmembrane (TM) regions; ii) highcalcium selectivity was likely to come at the price of complexity, andtherefore these were likely to be large proteins; iii) the high calciumselectivity of this type of channel was likely to be useful and,therefore, highly conserved; and iv) these channels should be expressedin one or more types of lymphocytes, since ICRAC is best defined inthose cell types. Since the full genome of the nematode C. elegans isnearing completion, and IP3-dependent calcium signals have recently beenshown to be required for one or more aspects of C. elegans development,we took the set of proteins encoded by this genome (at the time thissearch was initiated WORMPEP14 was the available predicted protein set)and began searching for proteins which fit the criteria above. Thissearch began by proceeding in alphabetical order through WORMPEP14 andarbitrarily excluding all proteins below approximately 1000 amino acidsin size, followed by focusing on remaining proteins with clear TMspanning regions similar to those of other calcium channels. We stoppedthis screen on encountering a protein designated C05C12.3, a predictedprotein of 1816 amino acids (SEQ ID NO:13). C05C12.3 was noteablebecause its central pore region had some sequence similarity to but wasclearly distinct from members of the Trp family of calcium channels, andthe hydrophobicity plot of this region showed a characteristically widespacing between the fifth and sixth TM regions for the amino acidresidues which are thought to line the channel pore region and mediatethe calcium selectivity of the channels. In addition, it lacked anyankyrin repeats in the region amino-terminal to its pore region, furtherdistinguishing it from other Trp family proteins.

We then used C05C12.3 for BLAST alignment screening of the rest of theC. elegans genome and also mammalian databases for homologous proteins,revealing two other C. elegans homologues (SEQ ID NO:14 and SEQ IDNO:15), and also a recently cloned mammalian protein named melastatin-1(MLSN-1/SOC-1, SEQ ID NOs:9 and 10, and GenBank Acc. No. AF071787).Using these sequences, we subsequently performed an exhaustive screeningof publicly accessible EST databases in search of lymphocyte homologues,but were unsuccessful in detecting any homologous transcripts in anylymphocyte lines. Since MLSN-1 (SEQ ID NOs:9 and 10) was expressedexclusively in melanocytes and retina by Northern blot hybridization andby EST database searching, there was no evidence that this type ofchannel was expressed in the type of cell in which ICRAC-like currentswere best defined. Subsequent BLAST searches picked up mouse ESTsequence A1098310 (SEQ ID NO:22) from a monocyte cell line. TheI.M.A.G.E. consortium clone containing the above-identified EST was thenpurchased from ATCC (clone ID. 1312756, Manassas, Va.) and was furthercharacterized. Using other portions of this sequence in EST searches, wesubsequently picked up similar sequences in human B-cells (SEQ ID NOs:20and 21), and other cell types as well (SEQ ID NOs: 11, 12, 16, 17, 18,and 19). Most of these sequences were subsequently identified to be partof the 3′-UTR or of the carboxy terminal region of the proteins, whichare not readily identifiable as Trp channels, providing an explanationfor the art's inability to detect any type of Trp related transcripts inlymphocytes. Partial sequences from the 5′ and/or 3′ ends of the aboveidentified clones were then used to screen leukocyte and kidney cDNAlibraries to extend the original sequences more toward the 5′ and/or 3′ends.

In view of the foregoing, it was concluded that channels of this typewere expressed in many types of lymphocytes, and therefore were membersof a new family of SOC/CRAC calcium channels.

Experimental Procedures

Screening of the cDNA Libraries

Leukocyte and kidney cDNA libraries from Life Technologies(Gaithersburg, Md.) were screened using the Gene Trapper II methodology(Life Technologies) according to manufacturer's recommendation, usingthe inserts of I.M.A.G.E. clone ID nos. 1312756 and 1076485 from ATCC(Manassas, Va.), under stringent hybridization conditions. Usingstandard methodology (Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., NewYork), individual cDNA clones were subjected to 3–4 rounds ofamplification and purification under the same hybridization conditions.

After excision from the vector and subcloning of inserts into theplasmid forms, several clones were sequenced by the Beth IsraelDeaconess Medical Center's Automated Sequencing Facility. Molecularbiological techniques such as restriction enzyme treatment, subcloning,DNA extraction, bacterial culture and purification of DNA fragments wereperformed according to methods well known in the art. Computer analysesof protein and DNA sequences was done using “Assemblylign” (OxfordMolecular, Cambell, Calif.). Multiple alignments of the SOC/CRAC familymembers were produced using the CLUSTAL facility of the MacVectorprogram. Restriction endonucleases, expression vectors, and modifyingenzymes were purchased from commercial sources (Gibco-BRL). Sequencingvectors for DNA were purchased from Stratagene (La Jolla, Calif.).

Once the first members of what appeared to be a novel family of calciumchannel receptors were identified and characterized, additional BLASTalignments were performed with the newly characterized nucleic acidsequences. An initial match was with genomic DNA fragment NH0332L11(Genbank Acc. No. AC005538). Using this genomic sequence, promers weredesigned and a number of cDNA libraries was surveyed by PCR. A prostatespecific message was identified and characterized, leading to theisolation and characterization of SOC-4/CRAC-3 (SEQ ID NOs: 31 and 32).

Functional Assays

Transient Expression of SOC/CRAC

In our initial transient expression experiments, we expressed or expectto express a SOC/CRAC molecule transiently in RBL-2H3 mast cells, JurkatT cells, and A20 B-lymphocytes using both electroporation and vacciniavirus-driven expression, and measured the calcium influx produced bydepletion of intracellular calcium stores with thapsigargin. Each of theforegoing techniques is well known to those of ordinary skill in the artand can be performed using various methods (see, e.g., Current Methodsin Molecular Biology, eds. Ausubal, F. M., et al. 1987, Green Publishersand Wiley Interscience, N.Y., N.Y.). Exemplary methods are describedherein.

Depletion of intracellular calcium stores is accomplished by treatingthe cells with 1 micromolar thapsigargin; alternative agents whichfunction to deplete intracellular stores are described in by Putney, J.W. Jr., in Capacitative Calcium Entry, R.G. Landes Co. and Chapman &Hall, 1997 and include, for example, ionomycin, cyclopiazonic acid, andDBHQ.

Calcium influx is determined by measuring cytoplasmic calcium asindicated using the fura-2 fluorescent calcium indicator (see, e.g., G.Grynkiewicz, M. Poenie, R. Y. Tsien, A new generation of Ca2+ indicatorswith greatly improved fluorescence properties, J. Biol Chem 260, 3440–50(1985), and M. Poenie, R. Tsien, Fura-2: a powerful new tool formeasuring and imaging [Ca2+]i in single cells, Prog Clin Biol Res 210,53–6 (1986)).

Patch Clamp Analysis and Determining Selectivity of SOC/CRAC

Patch clamp analysis of cells injected with SOC/CRAC cRNA is performedby using the general patch technique as described in Neher, E., “Ionchannels for communication between and within cells”, Science, 1992;256:498–502. Specific techniques for applying the patch clamp analysisto RBL cells are described in Hoth, M., and Penner, R., “Depletion ofintracellular calcium stores activates a calcium current in mast cells”,Nature, 1992; 355:3535–355. Additional protocols for applying the patchclamp technique to other cell types are described in Putney, J. W. Jr.,in Capacitative Calcium Entry, R.G. Landes Co. and Chapman & Hall, 1997

An exemplary protocol for patch clamp analysis of SOC/CRAC moleculeexpressed in RBL-2H3 mast cells using a recombinant vaccinia virus is asfollows. The currents elicited by store depletion are determined usingthe whole cell configuration (Neher, E., Science 1992; 256:498–502).Currents in SOC/CRAC expressing cells are compared to currents incontrol cells expressing an irrelevant protein or a classic Trp familycalcium channel known as VR1 (M. J. Caterina, et al., The capsaicinreceptor: a heat-activated ion channel in the pain pathway [seecomments], Nature 389, 816–24 (1997)) in order to assess thecontribution of SOC/CRAC expression. In addition, the magnitude of wholecell currents in the presence of extracellular calcium (10 mM), barium(10 mM), or magnesium (10 mM) are compared to determine the relativepermeability of the channels to each of these ions (Hoth, M., andPenner, R., Nature, 1992; 355:3535–355) and, thereby, determine theionic selectivity.

Pharmacologic Behavior of SOC/CRAC

For analysis of the pharmacologic behavior of a SOC/CRAC molecule, aSOC/CRAC molecule is expressed in RBL-2H3 mast cells using a recombinantvaccinia virus, and the degree of calcium influx elicited by storedepletion is monitored using a bulk spectrofluorimeter or a fluorescencemicroscope and the calcium sensitive dye fura-2 (G. Grynkiewicz, M.Poenie, R. Y. Tsien, A new generation of Ca2⁺ indicators with greatlyimproved fluorescence properties, J Biol Chem 260, 3440–50 (1985) and M.Poenie, R. Tsien, Fura-2: a powerful new tool for measuring and imaging[Ca2+]i in single cells, Prog Clin Biol Res 210, 53–6 (1986)). The levelof cytoplasmic calcium in SOC/CRAC expressing cells is compared to thelevel achieved in control cells expressing an irrelevant protein or aclassic Trp. family calcium channels known as VR1 (M. J. Caterina, etal., The capsaicin receptor: a heat-activated ion channel in the painpathway [see comments], Nature 389, 816–24 (1997)). These cells then arepre-incubated with the desired pharmacologic reagent, and again theresponse to store depletion is monitored. Comparison of the effect ofdepleting stores in SOC/CRAC expressing cells relative to controls inthe presence or absence of the pharmacologic reagent is used to assessthe ability of that reagent to modulate SOC/CRAC activity. Sphingosineis an exemplary molecule that can be used as pharmacologic reagents forpharmacologic characterization of SOC/CRAC calcium channels. See, e.g.,Mathes, C., et al., Calcium release activated calcium current as adirect target for sphingosine, J Biol Chem 273(39):25020–25030 (1998).Other non-specific calcium channel inhibitors that can be used for thispurpose include SKR96365 (Calbiochem) and Lanthanum.

Bulk Calcium Assays

Bulk calcium assays can be performed in a PTI Deltascan bulkspectrofluorometer using fura-2 as described in Scharenberg A M, et al.,EMBO J, 1995, 14(14):3385–94.

Gene Targeting

The method (and reagents) described by Buerstedde μM et al, (Cell, 1991,October 4;67(1):179–88), was used to generate “knockouts” in cells.Briefly, part of the chicken SOC-2/CRAC-1 genomic sequence coding forthe transmembrane region was cloned utilizing the human sequence as theprobe in a chicken library screen. Chicken SOC-2/CRAC-1 clones wereisolated and characterized using standard methodology. The putative exonand domain arrangement of the chicken SOC-2/CRAC-1, is depicted inFIG. 1. The exons coding for TM5 (pore region) and TM6, were replacedwith promoter/antibiotic cassettes (see FIG. 1). These targeting vectorswere then used to target (and replace) the endogenous gene in DT-40cells (chicken B lymphocyte cells).

Results

Example 1 Transient Expression of SOC/CRAC

In the above-identified cell lines and using both of the foregoingexpression techniques, SOC/CRAC expression enhancesthapsigargin-dependent influx. In addition, SOC/CRAC expression alsoenhances the amount of intracellular calcium stores. That this effect islikely due to SOC/CRAC acting as a plasma membrane calcium channel canbe confirmed by producing an in-frame carboxy-terminal translationalfusion with green fluorescent protein followed by confocal microscopy,revealing that SOC/CRAC is expressed predominantly as a plasma membranecalcium channel.

Example 2 Patch Clamp Analysis

The biophysical characteristics of SOC/CRAC enhanced currents whenexpressed in Xenopus oocytes are determined. SOC/CRAC cRNA injection isable to enhance thapsigargin-dependent whole cell currents. In addition.SOC/CRAC does not alter the reversal potential of these currents and thedetermination of the P_(ca)/P_(Na) ratio shows that SOC/CRAC channelsare highly calcium selective.

Example 3 Pharmacologic Behavior of SOC/CRAC

The pharmacologic behavior of SOC/CRAC is evaluated as described above.SOC/CRAC-enhanced influx is inhibited by sphingosine in a manner that issubstantially the same as that of endogenous thapsigargin-dependentcalcium influx.

Example 4 Gene Targeting

Transfection of DT-40 cells with the foregoing targeting vectors,selection for antibiotic resistance, and screening, is collectivellyrefered to, herein, as a round of targeting. For the first round oftargeting SOC-2/CRAC-1, 18/24 clones with homologous recombination ofthe targeting construct into one of the endogenous SOC-2/CRAC-1 alleleswere obtained. On the second round of targeting (in order to target thesecond allele and therefore generate a homozygous SOC-2/CRAC-1 mutantcell), 0/48 clones were obtained. These results indicate that a “null”SOC-2/CRAC-1 mutation is detrimental to DT-40 cells, and thatSOC-2/CRAC-1 is required for cell viability.

TABLE I Nucleotide Sequences with homologies to SOC/CRAC nucleic acidsSequences with SEQ ID NOs and GenBank accession numbers: SEQ ID NO:9,AB001535, AI226731, H18835, AA419592, AA261842, AA419407, AA592910,D86107, AI098310, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759,AA932133, R47363, N31660, AC005538, AA654650, AA370110, AA313170,AA493512, AI670079, AI671853.

TABLE II Amino Acid Sequences with homologies to SOC/CRAC polypeptidesSequences with SEQ ID NOs and GenBank accession numbers: SEQ ID NO:10,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,AB001535, AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532,AA551759, AA932133, R47363, N31660, NP003298, CAB00861, NP002411,CAA92726, CAB05572.

All references, patents, and patent documents disclosed herein areincorporated by reference herein in their entirety.

What is claimed is presented below and is followed by a SequenceListing.

1. An isolated nucleic acid molecule, comprising: a nucleic acidsequence that encodes SEQ ID NO: 30, or a nucleic acid sequence that isfully complementary to the nucleic acid sequence that encodes SEQ ID NO:30.
 2. An isolated nucleic acid molecule comprising SEQ ID NO: 29, or anucleic acid molecule that is fully complementary to the nucleic acidmolecule comprising SEQ ID NO:
 29. 3. An isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:30.
 4. A pharmaceutical compositioncomprising: an agent comprising the isolated polypeptide of claim 3, anda pharmaceutically acceptable carrier.
 5. An isolated nucleic acidmolecule comprising nucleotides 73–3714 of SEQ ID NO: 29, or a nucleicacid molecule that is fully complementary to the nucleic acid moleculecomprising nucleotides 73–3714 of SEQ ID NO:
 29. 6. An isolated nucleicacid molecule comprising SEQ ID NO: 25, or a nucleic acid molecule thatis fully complementary to the nucleic acid molecule comprising SEQ IDNO: 25.